This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

1,5-Anhydro-D-fructose (AF) is a mono-saccharide directly formed from starch and glycogen
by the action of α-1,4-glucan lyase (EC 4.2.2.13). Our previous study has indicated
that AF increases glucose tolerance and insulin secretion in NMRI mice after administration
through a gastric gavage in a single dose at 150 mg per mouse. In this study, we used
high-fat feeding of C57BL/6J mice to examine the influence of long-term administration
of AF on glucose-stimulated insulin secretion in vivo and in vitro. We found that 8-weeks of high-fat feeding increased body weight, fasting blood glucose
and insulin levels in C57BL/6J mice when compared to mice fed normal diet. Impaired
glucose tolerance was also observed in mice receiving 8-weeks of high-fat diet. In
contrast, AF (1.5 g/kg/day), administered through drinking water for 8-weeks, did
not affect body weight or food and water intake in mice fed either the high-fat or
normal diet. There was no difference in basal blood glucose or insulin levels between
AF-treated and control group. Oral glucose tolerance test (OGTT) showed that AF did
not affect glucose-stimulated insulin secretion in mice. In in vitro studies with isolated islets, AF did not influence glucose-stimulated insulin secretion
in mice receiving either high-fat or normal diet. We therefore conclude that when
given through drinking water for 8 weeks at 1.5 g/kg/day, AF has no effect on glucose-stimulated
insulin secretion in C57BL/6J mice challenged with a high-fat diet.

Background

1,5-Anhydro-D-fructose (AF) is a mono-saccharide having structural similarity to glucose
[1]. It is produced by the degradation of starch and glycogen catalysed by the enzyme
α-1,4-glucan lyase [1]. AF is present in fungi and algae, including edible fungal and algal species [2,3], as well as in mammalian tissues including rat liver [1,4]. In vitro studies have indicated that enzymatic oxidation of 1,5-anhydro-D-glucitol (AG) by
fungal pyranose 2-oxidases results in the formation of AF; however, this reaction
has not been demonstrated in vivo in mammals [5]. In mammals, the further metabolism of AF involves a NADPH-dependent specific reductase
that reduces AF to AG [1,6]. It has been reported that AG, the second most abundant polyol after glucose in human
fluid, stimulated insulin secretion in two rodent insulinoma cell lines studied, i.e., rat RINr and mouse MIN6 at physiological relevant concentrations [7]. In fungi and red algae AF is metabolised to secondary metabolites such as microthecin,
ascopyrones and echinosporin [2,3,8,9]. However, the importance of AF in mammalian physiology remains elusive. The works
by Hisaku et al. [10], Fujisie et al. [11] and Yamaji et al. [12] have indicated that AF has antioxidant and antimicrobial effects, suggesting a potential
biological role for AF in mammals. Furthermore, we have previously shown that when
given through a gastric gavage (150 mg) together with glucose (150 mg/mouse), AF induces
glucose tolerance, insulin secretion and increases in plasma levels of glucagon-like
peptide-1 (GLP-1) [13]. The effect of AF on glucose tolerance, however, was not detected when administered
intravenously [13]. Based on these observations, the role of AF in increasing endogenous GLP-1 secretion
needs to be explored further to clarify the discrepancy. In the current study, we
used high-fat feeding of C57BL/6J mice as a model to investigate the effect of long-term
administration of AF on glucose-stimulated insulin secretion in vivo and in vitro. C57BL/6J mice are susceptible to high-fat diet and develop glucose intolerance more
readily than other strains [14]. Furthermore as indicated above, as AF metabolism is an energy-consuming process
due to the use of NADPH in its reduction to AG [1,6], feeding mice with AF might reduce the extent for obesity development.

Methods

Animal

Four-week old female C57BL/6J mice weighing 15 g were obtained from Bomholtgaard Breeding
and Research Center, Denmark. Animals were housed on a 12-h light/dark cycle with
ad libitum access to diets and water. The mice were fed with either a standard rodent food or
a high-fat diet (#D12310 and #D12309; Research Diets, New Brunswick, NJ). The normal
diet had a caloric density of 12.6 kJ/g and contained 25.8% protein, 62.8% carbohydrates
and 11.4% fat. The high-fat diet consisted of 16.4% protein, 25.6% carbohydrates and
58.0% fat with a caloric density of 23.6 kJ/g. The mice remained on each of the diets
for 8 weeks. During the 8 weeks, AF (1.5 g/kg/day) dissolved in tap water was made
accessible to the mice. The control group received tap water. The food and water intake
and body weight were recorded weekly.

Oral glucose tolerance test

After 8 weeks of AF treatment, blood was drawn from the intra-orbital bullar plexus
of all mice for the measurement of basal glucose and insulin levels. For oral glucose
tolerance test (OGTT), mice fasting overnight were given glucose (150 mg/mouse) orally
and their blood was collected at times 0, 15, 30, 60, 90 and 120 min following glucose
administration. All procedures using animals were approved by the local Ethics Committee
and followed the guidelines for experimentation in animals (European Economic Community
Council Directive 86/609/EEC).

Insulin secretion in vitro

Pancreatic islets were isolated from mice using the collagenase isolation technique.
Briefly, the common bile duct was ligated at the papilla vateri and cannulated after
a midline incision. The pancreas was filled with 3 ml of ice-cold Hank's balanced
salts (HBSS) supplemented with 0.4 mg/ml collagenase P (Roche Molecular Biochemicals,
Mannheim, Germany) before removal and then incubated at 37°C for 19 min. After washing
the incubated islets for three times with HBSS, they were handpicked under a stereomicroscope
and incubated overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum,
2.05 mmol/l L-glutamine, 2.5 μg/ml amphotericin B, 100 IU/ml penicillin and 100 μg/ml
streptomycin at 37°C in humidified air equilibrated with 5% CO2. After overnight incubation, the islets were washed three times and then pre-incubated
for 60 min at 37°C in a Hepes medium (pH 7.4 supplemented with 0.1% human serum albumin
(Sigma) and 3.3 mmol/l glucose. The Hepes medium consisted of 125 mmol/l NaCl, 5.9
mmol/l KCl, 1.2 mmol/l MgCl2, 1.28 mmol/l CaCl2 and 25 mmol/l Hepes. After pre-incubation, groups of three islets were transferred
into separate chambers containing 200 μl of the Hepes or RPMI medium supplemented
with glucose at various concentrations. Following incubation at 37°C for 60 min, 25
μl of the medium was collected and stored at -20°C until analysis.

Statistics

Mean values ± SE for glucose and insulin plasma levels are shown. Statistical comparisons
for differences between AF and control were performed by unpaired Student t test.

Results

AF treatment for 8 weeks

As seen in Table 1, 8 weeks of high-fat feeding increased body weight and blood glucose compared with
the mice fed normal diet. By contrast, AF did not change/reduce body weight, blood
glucose and insulin levels in mice fed high-fat diet. There was no difference in the
food and water intake levels between AF-treated and control group of mice fed high-fat
diet. In mice fed normal diet, AF did not affect body weight, food and water intake,
or fasting blood levels for glucose and insulin (Table 1).

Islet studies

Insulin secretion over a wide range of glucose concentrations was similar in isolated
islets from control and AF-treated high-fat-fed mice (Figure 3). Similar results were also observed in isolated islets from normal diet-fed mice
after 8 weeks of treatment with AF (data not shown).

Figure 3.Insulin secretion in isolated islets from AF treated C57BL/6J for 8 weeks. Glucose-stimulated insulin secretion was determined during a 60-min incubation of
islets isolated from C57BL/6J mice after 8 weeks of a high-fat diet. Results are expressed
as means ± SE for each group and glucose concentration (n = 4).

Discussion

Our previous results demonstrated that AF increased glucose tolerance and insulin
secretion in NMRI mice after being administered through a gastric gavage in a single
high dose of 75 or 150 mg per mouse. Furthermore, in these mice, AF enhanced the increase
in plasma levels of the gut hormone, GLP-1. In the present study, we examined whether
long-term administration of AF at a more moderately high dose influences glucose-stimulated
insulin secretion in C57BL/6J mice. C57BL/6J mice have been shown to be susceptible
to developing glucose intolerance more readily than other strains when maintained
on a high-fat diet [14-16] and were the selected model in the current study.

It was observed that AF did not influence glucose tolerance and insulin secretion
in C57BL/6J mice fed a high-fat diet (Figure 1, 2, 3). In this study, AF was administered to the mice, fed either a high-fat or normal
diet for 8 weeks, via drinking water. The dose of AF was 1.5 g/kg/day, or around 30
mg/mouse/day. This dose is much lower than the one used in our earlier single and
high dose study [13]. It would be possible that the current dose of 30 mg/mouse/day was not sufficient
to change glucose tolerance and insulin secretion. However, high dose of AF, which
might have an effect, may not have practical application either as a drug or a health
food additive. Beside the different mouse models and the high dose used [13], the routes of administration of AF were also different in these two studies. In
the previous study [13], AF was given through a gastric gavage tube placed in the stomach of the mice, while
AF was administered through drinking water in the present study. It is not sure if
these might have influenced the effective concentration of AF in the circulating blood,
which may be important for the biological activity of AF. On the other hand, if the
major in vivo physiological function of AF is by its antioxidant activity [12], it might not improve glucose tolerance under these conditions since it has been
reported that high fat does not cause oxidative stress [17]. It is also known that fatty acids acutely amplify glucose-induced insulin secretion
from the pancreatic β-cell, but they become harmful when present at elevated levels
for prolonged periods of time due to their deleterious effects on β-cell function
including inhibition of insulin secretion, the so-called lipotoxicity [17]. That is, the possible positive effect of AF on glucose tolerance might have been
blunted due to high-fat diet feeding. A high-fat diet has been shown to induce hyperglycemia,
hyperinsulinemia, hyperlipidemia, obesity and glucose intolerance in C57BL/6J mice
(Table 1) [15,16,18]. Thus, an additional drug might be needed together with AF in order to overcome the
strong changes in pathophysiology in C57BL/6J mice challenged with a high-fat diet
compared to acute administration of a single high dose of AF in normal NMRI mice [13]. It should be noted also that though AF is energy-negative feeding mice at 1.5 g/kg/day
apparently did not affect the weight gain (Table 1).

AF has recently been reported to have several health beneficial effects including
anti-inflammatory and anti-cancer effects [19-21] beside its antioxidant effect [12]. Though no indication of improved glucose tolerance was evident, this study further
confirmed our early observations [22,23] that AF is a safe sugar to consume as no adverse effect was observed in the current
study.

Conclusions

With C57BL/6J mice as model which is liable to develop glucose intolerance when fed
on a high-fat diet, 1,5-Anhydro-D-fructose (AF) at 1.5 g/kg/day, administered through
drinking water for 8-weeks, did not affect body weight gain or food and water intake
fed on either high-fat or normal diet. No difference in basal blood glucose or insulin
levels between AF-treated and control group was found. These indicate that AF as a
food antioxidant does not apparently interfere with sugar metabolism in mice.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

JM was the major person performed the experiments using the 1,5-anhydro-D-fructose
prepared and provided by SY. The experiments were designed and discussed by JM, SY
and BA. All these work were performed at Department of Medicine, Lund University,
Lund, Sweden.

Acknowledgements

We are grateful to Ms Lilian Bengtsson and Ms Lena Kvist for excellent technical assistance.
This work was supported by grants from European Commission "New Products from Starch
Derived 1,5-Anhydro-D-Fructose" (NEPSA) with the contract No. of QLRT-2000-02400 under
FP5.